Supplemental Methods
Six independent colonies from each of two E. coli lines (wild-type and yjeF knockout)
were grown overnight in M9 minimal medium (0.2% glucose) and used to inoculate 2 mL of
fresh medium to an optical density of 0.05 at 600 nm. Cultures were grown at 42°C with
shaking for 5-6 h until optical density reached 1.3±0.1 at 600 nm, then an equivalent of 1
mL culture at an optical density of 2.0 was collected in 1.5-mL Eppendorf tubes by
centrifugation and the pellet was frozen in liquid nitrogen. Samples were stored at -80°C.
Analysis by LC-MS was conducted on the supernatant from cell pellets extracted with 1
mL cold acetonitrile: isopropanol: water (3:3:2), dried overnight then resuspended with 30
L of acetonitrile:water (80:20) plus internal standards. Chromatography was performed
on an Agilent 1290 Infinity LC System (Agilent Technologies, Santa Clara, CA) using a
Waters Acquity 1.7 m BEH HILIC 150 x 2.1 mm UPLC column kept at 45oC. Mobile phase
consisted of water with 10 mM ammonium formate and 0.125% formic acid (A) and 95:5
acetonitrile/water with 10 mM ammonium formate and 0.125% formic acid (B); flow rate
was 0.4 mL/min throughout acquisition. The gradient method was 0 – 2min: 100% B, 2 –
7.7 min: 70% B, 7.7 – 9.5 min: 40% B, 9.5 – 10.25 min: 30% B, 10.25 – 12.75 min: 100% B,
12.75 – 16.75 min: 100% B. MS and MS/MS data were collected in positive and negative
polarity with a 4 spectra/s scan rate and mass calibration was maintained by constant
infusion of reference ions. Precursor ions were isolated with a narrow isolation width (~1.3
Da) and variable collision energy was applied based on precursor ion m/z for datadependent MS/MS data generation.
Data processing was performed using the open-source software MZmine 2 [43] and the
in-house MZmine post-processor (unpublished). Compound identification was based on
accurate-mass and MS/MS matching in the NIST MSPepSearch GUI
(http://peptide.nist.gov/) with precursor ion and fragment peak tolerance of 0.05 m/z units
using the NIST, Metlin, MassBank and LipidBlast libraries; additional MS/MS and retention
time matching to in-house libraries was also performed.
Database API is implemented using US Department of Energy Biological Knowledge
Base JSON Remote Procedure Call framework and is available at
https://bitbucket.org/jgj333/mine-server. The MINE web application is built with
AngularJS and available at https://bitbucket.org/jgj333/mine-website.